Flowers having new traits are always considered to have high value in the flower industry. The development of plants capable of inducing a change in color, considered to be the most important trait among flowers, is viewed with particular importance, and various colors of flowers have been developed thus far through selective breeding using classic crossbreeding techniques. Although crossbreeding is an effective method of selective breeding, since plants are subjected to their own unique genetic limitations, they have the shortcoming of only allowing the use of the genetic resources of related species able to be crossbred therewith. For example, despite having been crossbred for many years, violet to blue roses, carnations, chrysanthemums and lilies, bright red gentians and irises, and yellow morning glories have yet to be produced.
Flower color is attributable to four types of pigments consisting of flavonoids, carotenoids, chlorophylls and betalains. Among these, flavonoids exhibit a diverse range of colors in the manner of yellow, red and blue. The group of flavonoids exhibiting red, violet and blue colors are generically referred to as anthocyanins, and the structural diversity of anthocyanins is one of the reasons for the diverse range of flower color. When considering the biosynthetic pathway thereof, anthocyanins can be broadly classified into three groups according to their aglycone structure. Pelargonidin-type anthocyanins are frequently contained in flowers having bright red color in the manner of carnations and geraniums, while delphinidin-type anthocyanins are frequently contained in flowers having blue or violet color. The reason for the absence of blue or violet varieties among roses, carnations, chrysanthemums and lilies is that these plants do not have the ability to synthesize delphinidin-type anthocyanins.
In addition to the accumulation of delphinidin, any of the following other factors are considered to be required to allow flowers to have blue color: (i) the anthocyanin must be modified by one or a plurality of aromatic acyl groups, (ii) the anthocyanin must be present together with a copigment such as flavone or flavonol, (iii) iron ions or aluminum ions must be present together with the anthocyanin, (iv) the pH of vacuoles in which the anthocyanin is localized must rise from a neutral pH to a weakly alkaline pH, or (5) the anthocyanin, copigment and metal ions must form a complex (and such anthocyanins are referred to as metalloanthocyanins) (refer to Non-Patent Document 1).
Considerable research has been conducted on flavonoid and anthocyanin biosynthesis, and related biosynthetic enzymes and genes encoding those enzymes have been identified (refer to Non-Patent Document 2). For example, the gene for flavonoid-3′,5′-hydroxylase (F3′5′H), which hydroxylates the flavonoid B ring required for biosynthesis of delphinidin, is obtained from numerous plants. In addition, by introducing these F3′5′H gens into carnations (refer to Patent Document 1), roses (refer to Non-Patent Document 3 and Patent Documents 2 and 3) or chrysanthemums (refer to Patent Document 4), a genetically modified plant is produced in which delphinidin accumulates in the flower petals thereof and flower color changes to blue (refer to Non-Patent Document 4). Such carnations and roses are available commercially.
Flavones are a type of organic compound in the form of cyclic ketones derived from flavans, and in the narrow sense, refer to 2,3-dehydroflavan-4-one, a compound represented by the chemical formula C15H10O2 and having a molecular weight of 222.24. In the broad sense, derivatives belonging to flavones are generically referred to as “flavones”. Flavones in the broad sense (flavones) constitute one category of flavonoids, and those flavonoids having a flavone structure for the basis skeleton thereof and not having a hydroxyl group at position 3 are classified as “flavones”. Typical examples of “flavones” include apigenin (4′,5,7-trihydroxyflavone) and luteolin (3′,4′,5,7-tetrahydroxyflavone). In the description of the present application, the term “flavones” refers to flavones in the broad sense, namely derivatives belonging to flavones.
Genes for flavone synthases (FNS) required for biosynthesis of flavones are obtained from numerous plants. Flavones are known to have the effect of producing the deep blue color of anthocyanins when in the presence of anthocyanins, and these FNS genes are attracting attention in the field of flower color modification. As a result of introducing an FNS gene into a rose not having the ability to synthesize flavones together with F3′5′H, simultaneous to accumulation of delphinidin in flower petals, flavones also accumulate therein causing flower color to change to an even bluer color (refer to Patent Document 5). In addition to producing a blue flower color, since flavones also absorb ultraviolet rays, they have the function of protecting plants from ultraviolet rays or serving as a visual signal for insects in the case of insect-pollinated flowers. In addition, flavones are also involved in interaction between plants and soil microorganisms. Moreover, flavones are also used as ingredients of foods and cosmetics as components that are beneficial for health. For example, flavones are said to have an anticancer action, and the ingestion of foods containing large amounts of flavones has been demonstrated to treat or prevent cancer.
In addition, genes that modify anthocyanins and flavones are obtained from numerous plants. Although examples thereof include glucosyltransferases, acyltransferases and methyltransferases, glucosyltransferases (GT) that transfer glucose to the hydroxyl group at position 3 of anthocyanins are described here. For example, genes that encode proteins having activity that transfers glucose to the hydroxyl group at position 3 of an anthocyanin have been isolated from such plants as gentians, perillas, petunias, roses or snapdragons (refer to Non-Patent Documents 4 to 6 and Patent Document 6). Genes that encode proteins having activity that transfers glucose to the hydroxyl group at position 5 of an anthocyanin have been isolated from such plants as perillas, petunias, gentians, verbenas or torenias (refer to Non-Patent Documents 5 to 7 and Patent Document 7). Genes that encode proteins having activity that transfers glucose to the hydroxyl group at position 7 of a flavone have been isolated from thale cress (refer to Non-Patent Document 8). A gene that encodes a protein having activity that transfers glucose to the hydroxyl group at position 7 of baicalin has been isolated from baical skullcap, and a protein obtained by expressing this gene in Escherichia coli (E. coli) has been reported to catalyze a reaction that demonstrates activity that transfers glucose to the hydroxyl group at position 7 of a flavonoid (refer to Non-Patent Document 9). Genes that encode a protein having activity that transfers glucose to the hydroxyl group at position 3′ of an anthocyanin have been isolated from gentians, butterfly peas and florist's cineraria (refer to Patent Document 8). In addition, a gene that encodes a protein having activity that sequentially transfers glucose to hydroxyl groups at two different locations on the A ring and C ring of an anthocyanin has been isolated from roses (refer to Patent Document 9). A gene that encodes a protein having activity that sequentially transfers glucose to two different locations on the B ring of an anthocyanin has been isolated from butterfly peas (refer to Patent Document 10).
Although the aforementioned glucosyltransferases use UDP-glucose as a glycosyl donor, glucosyltransferases have recently been identified that use acyl-glucose as a glycosyl donor. A gene that encodes a protein having activity that transfers glucose to the hydroxyl group at position 5 of anthocyanidin 3-glucoside has been isolated from carnations, while a gene that encodes a protein having activity that transfers glucose to the hydroxyl at position 7 has been isolated from larkspur (refer to Non-Patent Documents 10 and 13). Moreover, a protein obtained by expressing a glucosyltransferase gene derived from Livingstone daisies has been reported to demonstrate activity that transfers glucose to either of the hydroxyl groups at position 4′ or position 7′ of a flavonoid in vitro (refer to Non-Patent Document 11). In addition, a polynucleotide that encodes a protein having activity that transfers a sugar to the hydroxyl group at position 4′ of a flavone has been isolated from Nemophilas (refer to Patent Document 11).
In this manner, although there are numerous glucosyltransferases in the form of proteins having activity that transfer glucose to various hydroxyl groups, there are still thought to be a large number of glucosyltransferases for which the function thereof has yet to be identified. Thus, there continues to be a need to acquire glucosyltransferases that function in plants and are useful for modifying flower color.